Method detecting antigens and/or antibodies

ABSTRACT

A suspension of inert particles is prepared in an aqueous solution, to which an antibody or an antigen and a carrier-bound antigen or antibody, respectively, are added in any desired order. After centrifuging, the positive, weakly positive, or negative reaction can easily be recognized on the basis of a simple pattern.

This is a divisional application under 37 C.F.R. §1.60 of pending priorapplication Ser. No. 07/969,532 filed on Oct. 30, 1992, now U.S. Pat.No. 5,338,689 which is a continuation of Ser. No. 07/684,459 filed Apr.11, 1991 (now abandoned), which is a continuation of Ser. No.07/122,152, filed Nov. 11, 1987 (now abandoned).

This invention relates to biological test methods, and more particularlyto a method of detecting antibodies or antigens, of the type whereincomplexes of carrier-bound antibodies with antigens or of carrier-boundantigens with antibodies in an aqueous medium are made opticallyvisible, using inert particles such as polyacrylamide gel particles. Theinvention further relates to test kits for detecting antigens and/orantibodies according to the aforementioned method.

Carrier-bound antigens and antibodies are routinely utilized for amultitude of analytical determinations. In this connection, adistinction is generally made between two principles:

(a) The antigen or antibody is bound to a solid carrier, such as smallglass or plastic test tubes, glass or plastic beads, glass or plasticplates, paper, etc., and the liquid containing the antibody or antigento be identified is added. If, after a certain reaction time, the latterreact with the bound antigen or antibody, they are bound to the carrierand can, at least by means of a further reaction, be made measurable. Asa rule, this takes place by labeling the antibody or antigen withradioactive, fluorescent, or enzymatically active tagging substances. Adrawback of this method is that it is trouble-prone. Since washing isnecessary after each reaction, insufficient washing or insufficientremoval of the washing solution can falsify the results. Moreover, thismethod is hardly applicable to natural, carrier-bound antigens orerythrocytes, leukocytes, platelets, and other natural cells because oftheir size.

(b) Another principal method consists in binding the antigen or antibodyto small particles such as latex or erythrocytes. After a certainreaction time with the antibody or antigen to be identified, theevaluation takes place on the basis of the agglutination pattern. Unlikethe method described under (a) above, antigens and antibodies onerythrocytes, leukocytes, or platelets can be identified by this method.Drawbacks of this method are that, especially in a weak concentration,it is hard to differentiate between agglutinated and non-agglutinated(free) particles. Moreover, free particles can easily be absorbed on thereaction vessel or the agglutination pattern mechanically destroyed. Afurther source of error resides in the possible adhesion of freeparticles to one another, thus simulating an agglutination. All theseeventualities can lead to a false evaluation.

European Published Application No. 0 039 195 describes anantibody-detection method wherein erythrocytes in negatively chargedform are used in an isotonic solution of low ionic strength. A polymerin solution is added as an aid to agglutination. The agglutination isobserved visually, preferably under a microscope. The agglutinate can bedissociated again by adding a solution of citrate and sugar.

It is an object of this invention to provide an improved method ofdetecting antibodies or antigens which is simpler than the prior artmethods and free of their drawbacks.

Another object of this invention is to provide such a method in whichreadings can be taken easily and reliably.

To this end, in the method according to the present invention, asolution containing an antibody or an antigen is brought into contactwith a carrier-bound antigen or antibody, respectively, a slurry orsuspension of inert particles being added before, during, or after thisreaction; upon formation of an antigen-antibody complex, the latterlying upon the sediment of the inert particles in the strongly positivecase and being present within the inert particles in the weakly positivecase; and in the absence of an antigen-antibody complex, i.e., in thenegative case, the carrier-bound antibodies or antigens lying beneaththe inert particles sediment.

The test kit according to the present invention contains at least onereaction vessel, inert particles, and one carrier-bound antibody and/ orantigen per reaction vessel.

The chemical composition of the inert particles used in the method isnot critical. The term "inert" is intended to mean that the particlesmust not enter into any unspecific reactions with the antigens orantibodies. Preferably, such inert porous particles as are available incommerce for liquid or gas chromatography will be used. These areproducts on the basis of cross-linked polymers, such as agarose,polyacrylamide, polydextran, or styrene-divinylbenzene polymers (e.g.,"Sephadex," "Sepharose," or "Sephacryl," sold by Pharmacia AB, Uppsala,Sweden; or "Biogel," sold by Bio-Rad Laboratories, Richmond, Calif.).Porous glass or silica gel also enters into consideration. The particlesize is preferably from 10-200 microns. Those skilled in the art candetermine by means of simple preliminary tests whether the particles areusable for a certain method of determination. As in the base of theabove-mentioned inert particles, the type of carrier for the antigen orantibody is also not critical. For visual or optical-automatic measuringmethods, it should be colored by nature (e.g., erythrocytes). However,the carriers may also be tagged by means of a stain (e.g., latex,polymerized agarose). Other marking systems also enter intoconsideration, such as isotope, fluorescent, or enyzme labeling. Thesenaturally require an appropriate measuring method. Moreover, thecarriers are preferably likewise in particle form, the antigens orantibodies being bound to their surfaces.

The antigens and antibodies are preferably bound to these particles bychemical bonding, the type of bonding not being critical. Certainantigens, such as those of erythrocyte and platelets, already existbound to these carriers. Leukocytes and platelets may be stained byknown methods, if need be.

In the case of leukocytes or platelets, the inert particles mayalternatively be stained. In that even, the reading zone is whitish, andthe inert particles are colored.

According to the method of the present invention, it is possible todetermine the free antigens or antibodies, certain carrier-boundantibodies or antigens, respectively, having to be given. Conversely,the carrier-bound antigens or antibodies may also be determined ,thefree antigens or antibodies, respectively, having to be given. Thereaction vessels used for carrying out the method are, as a rule, smalltest tubes or microtiter plates of glass or plastic. The material is notcritical. The small test tubes may be either rounded or pointed, theshape being chosen as a function of the reading technique and thetechnical separation method and the quantity of material.

For carrying out the method of the present invention, small tubes ormicroreaction vessels are preferably used. Any desired number of thesemay, for example, be arranged side by side on a card. There may be anadditional vessel, e.g., for preparing a test dilution or testsuspension. The vessels and cards may be of plastic, such as PVC/PVDC,PET, or polystyrene. The vessels may, for instance, be made by theblister method, by a welding process, or by gluing. The vessels maycontaining the inert particles or industrially prepared reactionsolutions. Since the invented method preferably includes a centrifugingstep, special, suitable centrifuge heads must be used for the tubes orthe cards with the containers. With one advantageous centrifuge head, atleast 12 cards having, for example, six reaction vessels can becentrifuged simultaneously. In this case, 72 tests can be carried outparallel to one another.

Although sedimentation can be brought about by allowing the vessels tostand and taking advantage of the force of gravity, it is moreadvantageous to use the centrifuging method since the desiredsedimentation can be achieved after only a short time. The optimumconditions (centrifuging a time and number of g) must be ascertained foreach analysis system since the specific gravity, size, shape,deformability, and stability of the carrier-bound antigen-antibodycomplex, or the non-complexed, carrier-bound antibodies and antigens,and of the inert particles have an influence which is difficult tocalculate.

Certain aspects of the invention will now be described in detail withreference to the accompanying drawings, in which:

FIGS. 1(a)-(c) are diagrammatic elevations of three test tubes showingthe patterns occurring in positive, weakly positive, and negativereactions,

FIG. 2 is a side elevation and a front elevation of a typical microtubesuitable for affixing to a card,

FIGS. 3a and 3b are a side elevation and a front elevation,respectively, of a card with six small test tubes as in FIG. 2,

FIG. 4 is a diagram of the way in which the test is carried out, and

FIGS. 5, 6, and 7 are elevations of test cards for typical routineclinical tests.

Theoretically, the method according to the present invention can be usedto test a sample of liquid for an antigen or an antibody which isspecific to a known antibody or antigen, respectively. Either the knownantigen or antibody, or the unknown antibody or antigen, must be boundto a carrier, e.g., an erythrocyte. The method of detection is basedupon the realization that carrier-bound antigens and antibodies havedifferent centrifugation properties from carrier-bound antigen-antibodycomplexes. If an antigen-antibody complex on a carrier is centrifugedtogether with a suspended, inert carrier material, the carrier-boundcomplex lies on the inert particles. If no reaction has taken place,only a free, carrier-bound antigen or antibody is in the test tube withthe inert, suspended material. After centrifuging, this antigen orantibody is situated beneath the layer of inert particles. In this way,the positive or negative antibody reaction can plainly be read visually.It is also possible to automate this reaction. Weakly positive reactionsmay also occur, in which case the carrier-bound antigen-antibody complexis situated within the layer of inert particles.

A pattern of a positive reaction is shown in FIG. 1(a). If the reactionis only weakly positive, i.e., if only a few antigen-antibody complexesare formed, the antibody can be detected in the upper part of the inertparticles in the centrifuge glass; cf. FIG. 1(b). If, on the contrary,there is no antigen-antibody complex but only a carrier-bound, freeantigen or antibody, the latter is deposited beneath the inert particlesafter centrifuging. This is shown in FIG. 1(c).

As already mentioned, although various types of reaction vessels may beused, one such as is depicted in FIG. 2 is preferred. A side view isshown on the left, and a front view on the right. This small tube iscovered, thus making it possible for certain industrially manufacturedreagents to be supplied directly in the small test tube. The test tubeis suitable for affixing to a card, which may hold several tubes, e.g.,as shown in FIG. 3, where there are six small test tubes. FIG. 3a is aside view of the test card, while FIG. 3b is a front view. By means ofthis arrangement, a direct comparison of parallel tests is madepossible.

The test card may be made in various ways. For example, small tubes maybe glued to a card or may form an integral part of the card, in themanner of blister packaging. A mixture of inert particles and antibodiesor antigens may be hermetically enclosed in these tubes in apredetermined quantity by the manufacturer, in which case the tubes maybe sealed by means of a welded-on film. Test kits manufactured in thisway are easily handled and may be used in an automatic analysis method.In this case, the pipetting of specimens, identification, automaticreading, evaluation, print-out, etc., is controlled by means ofelectronic data-processing. A further advantage is that only very smallspecimens are necessary. For instance, with 10-50 μl of blood, alclinically relevant antigens can be detected with small amounts ofreagents. Microbatches are particularly important in the case ofsubstances which cannot be prepared synthetically and are available onlyin limited quantities; e.g., with 1 ml of the Rh antibody C, 20 antigendeterminations can be carried out by a conventional method, whereas 1000determinations are possible with the present method. If the test systemis appropriately prepared, the determinations are so easy to carry out,and the results so plainly readable, that the test can even be carriedout by auxiliary medical personnel.

Since no special laboratories are necessary, the interval betweendiagnosis and the start of therapy can be considerably reduced.

The invention will now be explained in more detail by means of thefollowing examples.

I. Blood Group Antigens (ABO System)

Example 1 -- A Antigen

(a) Preparation of inert particles suspension:

Five ml of "Sephacryl," S200 Gel (Pharmacia) is washed twice in salinesolution. After centrifuging (5 min., 1250 g), the supernatant isdiscarded, and the sediment is filled up to 4.5 ml with isotonicimidazol buffer (0.01 mol/l of imidazol 0.85% NaCl), pH 7.6.

(b) addition of antibody:

Five-tenths ml of anti-A is added to 4.5 ml of the above suspension. Thesuspension is mixed well and is ready for use in this form.

(c) Preparation of reaction vessels:

The above antibody suspension is placed in polyethylene microtubes(ET-29MM, sold by Miullian SA, Geneva, Switzerland), 100 μl in eachtube. The inert particles settle to the bottom of the tube within a fewminutes.

(d) Test procedure:

Twenty μl of an app. A% erythrocyte suspension of the unknown bloodspecimen in isotonic imidazol buffer, pH 7.0 (one part blood to nineparts buffer) is placed in the reaction vessel filled with the antibodysuspension and centrifuged for 10 min. at app. 100 g (Digifuge GL 122089 centrifuge, 800 rpm, sold by Heraeus, Hanau, West Germany).

(e) Evaluation:

If the tested blood belongs to blood groups A (A₁ or A₂), theantigen-antibody complex lies upon the inert particles (FIG. 1a). If itbelongs to the rare A subgroups A₃ or A_(x), the complex is distributedbetween the inert particles (FIG. 1b). If the blood belongs to groups Bor O, no agglutination can take place, and the erythrocytes collect atthe bottom of the tube beneath the inert particles after centrifugation(cf. Table 1).

                  TABLE 1    ______________________________________    Blood Group             A.sub.1 /A.sub.2 /A.sub.1 B/A.sub.2 B                           A.sub.3 /A.sub.3 B/A.sub.x                                       B/O    ______________________________________    Test     Positive      Weakly positive                                       Negative             (FIG. 1a)     (FIG. 1b)   (FIG. 1c)    ______________________________________

with anti-B instead of anti-A. The H antigen can be detected with Hlectin. A₁ is distinguished form A₂ by means of A₁ lectin. Example 1admits of a multitude of variations of the inert particles, the bufferused, the reaction vessels, the centrifugation time, and the number ofg. The origin of the antibodies--whether human, animal, or vegetable,whether polyclonal or monoclonal--is unimportant, provided thevariations do not change the reaction image (FIG. 4).

II. Rh-System Blood Groups (D, Du, C, E, c, e, Cw)

Example 2 -- E Antigen

The preparation of the reaction vessels (steps a-c) is as in Example 1.Instead of anti-A, anti-E is used, for example.

(d) Test procedure:

Step (d) of Example 1 is modified in that an enzyme solution ("DiaBrom,"sold by DiaMed AG, Murten, Switzerland) is used instead of the imidazolbuffer. It is known that concealed Rh antigens can be exposed thereby.Fifty μl of blood is suspended in 0.5 ml of enzyme solution. After app.5 min., 20 μl of this suspension is placed in the reaction vesselprepared as in steps (a)-(c) of Example 1 and centrifuged for 10 min. at100 g.

(e) Evaluation:

If the blood specimen contains antigen E, the antigen-antibody complexrests upon the inert particles (Table 2). Is antigen E not present, theerythrocytes collect under the inert particles.

                  TABLE 2    ______________________________________           E antigen present                      E antigen absent    ______________________________________    Test     Positive (FIG. 1a)                          Negative (FIG. 1c)    ______________________________________

The other Rh antigens can be determined completely analogously by meansof the appropriate antisera. The clinically important D-variant Du canbe differentiated by varying the concentration of anti-D.

III. Antibodies

Example 3 -- Reverse Test (Isoagglutinins)

The reaction vessels are prepared as in Example 1 (steps a-c), exceptthat imidazol buffer is substituted for the antibody.

(d) Test procedure:

Since antibodies are to be identified in this example, erythrocytes withknown antigens are used, e.g., A, B, and O test erythrocytes. These aresuspended in the known LISS solution (50 μl of blood and 2.0 ml ofLISS). Fifty μl of serum or plasma of the unknown blood specimen areplaced in each of three identically filled reaction vessels. One hundredμl of the A-cell suspension in LISS is added to the first tube, 100 μlof the B-cell suspension to the second tube, and 100 μl of the O-cellsuspension to the third tube. As in Examples 1 and 2, the suspensionsare centrifuged for 10 min. at 100 g.

(e) Evaluation:

If the unknown specimen contains anti-A, the first tube is positive, thesecond and third tubes negative (Table 3).

                  TABLE 3    ______________________________________    A cells          B cells   O cells    ______________________________________    Test   Positive      Negative  Negative           (FIG. 1a)     (FIG. 1c) (FIG. 1c)    ______________________________________

If the specimen contains anti-B, the second tube is positive, the firstand third negative. If the third tube is positive, and the first andsecond are positive or negative, the specimen contains antibodies whichare not anti-A and anti-B but directed to other antigens which lie onthe O cells. In this event, further investigation is necessary.

Example 4 -- Antibody Screening Procedure (Coombs Test)

The preparation of the reaction vessels is as in Examples 1-3, exceptthat Coombs serum (DiaMed AG) is added to the inert particlessuspension. As is well known, Coombs serum consists of anti-human-IgGand anti-complement C3 (C3b + C3d), as well as anti-IgM and anti-IgA,and is used for detecting and identifying, in serum from patients,antibodies directed to erythrocyte antigens. It is expedient first tocarry out an antibody screening procedure in which O erythrocytes havinga known antigen for clinically relevant antibodies are used. If theresult is positive, identification by means of a cell panel takes place.

Test procedure:

Fifty μl of serum or plasma from the patient is placed in each well orfunnel of one or more identical reaction vessels filled withCoombs-serum suspension. One hundred μl of the O-cell suspension (50 μlof blood in 2.0 ml of LISS) is added. The mixture is incubated for from10-20 min. at 37° C., at room temperature, or at from 2°-8° C.,depending upon the antibody sought, and centrifuged for 10 min. at 100g. Reading takes place as in the previous examples.

If the specimen contains an antibody against one or more erythrocyteantigens, the positive pattern of FIG. 1(a) appears. With weakerantibodies, the pattern is as in FIG. 1(b). Analogous to the above testprocedure, the antibody discovered may be identified by means of a cellpanel containing various antigens (e.g., products obtainable in commercefrom Ortho, Raritan, N.J.; Dade, Baxter, Miami, Fla.; and DiaMed).

IV. Blood-Typing on a Card

Example 5 -- Blood Group A, R₁ R₁ (CCDee), Kell Negative, ControlNegative

Typing can be done individually in small tubes, on microtiter plates, oron cards. In this example, blood-typing on cards designed fordetermining a patient's blood group is described. The preparation of thereaction vessels takes place as described above. The cards are shown inFIGS. 5-7.

V. Determination of Free Antigens or Antibodies Not Belonging toBlood-Group Systems

The reaction in Examples 1-5, in which the antigen is bound to theerythrocytes by its nature, can be applied to free antigens orantibodies by binding the respective antibodies or antigens to fixederythrocytes or other particles by known methods.

Example 6 -- Rheumatoid Factor Test

(a) Preparation of Erythrocytes:

Five ml of goat's blood in 0.011 mol citrate buffer, pH 6, is washedthree times in saline solution. The sediment is suspended with 5 ml ofsaline solution, mixed with 0.5 ml of 30% glutaraldehyde solution (E.Merck AG, Darmatadt, West Germany), and allowed to react for 24 hrs. atroom temperature with stirring. The sediment is washed three times insaline solution, mixed with 0.5 ml of rabbit IgG (10 mg/ml), andincubated for 24 hrs. at room temperature with stirring. After washingthree times in saline solution, an app. 40% suspension of the ladederythrocytes in imidazol buffer is prepared.

(b) Test procedure:

The reaction vessels are prepared as in Example 3. An app. 4%erythrocyte suspension is prepared with imidazol buffer. As in Example4, 50μl of serum from the patient is placed in the well of the testtube, and 20 μl of the 4% erythrocyte suspension is added. After about10 min. incubation at room temperature, centrifuging takes place asusual. If the serum contains rheumatoid factors, the reaction pattern ofFIG. 1(a) or (b) appears. If it is desired to ascertain the titer, thetest is repeated with serum diluted accordingly (0.9% NaCl).

The foregoing test is naturally not limited to the rheumatoid factors;for example, hepatitis antigens could be detected after coupling ofHBsAg antibodies. By coupling inactivated HIV virus or syntheticallyproduced proteins thereof to the erythrocytes, HIV antibodies canlikewise be easily detected.

Based upon these examples, it is immediately apparent that otherproteins, viruses, or bacteria, or antibodies thereof, can easily bedetermined by the same method.

What is claimed is:
 1. A method of detecting target antibodies orantigens by reaction with specific binding partners thereto, wherein onebut not both of the target or the binding partner is bound to a carrier,wherein a complex of the carrier-bound target and the binding partner,or of the target and the carrier-bound binding partner, forms anagglutinate which is optically detectable, said methodcomprising:providing a microreaction vessel containing a slurry orsuspension of inert particles and the known target or binding partnerthereto; adding to the vessel about 10-150 μl of a solution containingthe carrier-bound target or binding partner to allow formation of atarget-binding partner complex which is carrier-bound and opticallydetectable; centrifuging the vessel; and observing the location of thecarrier to determine the presence of the target antibody or antigen tobe detected, with a strongly positive reaction being indicated by thecarrier lying upon or in a top portion of a layer of the inertparticles, and a negative reaction being indicated by the carrier-boundtarget or carrier-bound binding partner lying beneath the inertparticles.
 2. A method of detecting analyte antibodies or antigens in afluid sample, by reaction with known antigen or antibody specificbinding partners thereto, respectively, wherein one, but not both, ofthe analyte or known antigen or antibody is bound to a carrier, saidmethod comprising:providing a microreaction vessel containing a slurryor suspension of inert particles and anti-immunoglobin; adding to thereaction vessel a fluid sample of about 10-150 μl which may contain theanalyte; adding to the vessel about 10-150 μl of a solution containingthe carrier-bound antigen or antibody to allow formation of an opticallydetectable complex between a) the anti-immunoglobin, and b) thecarrier-bound antigen and the analyte antibody, or the carrier-boundantibody and the analyte antigen; centrifuging the vessel; and observingthe location of the complex to determine the presence of the analyteantibody or antigen, with a strongly positive reaction being indicatedby the complex lying upon or in a top portion of a layer of the inertparticles, and a negative reaction being indicated by the absence of acomplex, with the carrier-bound antigen or antibody lying beneath theinert particles.